Water soluble prodrugs of hindered alcohols

ABSTRACT

The present invention is directed to novel water-soluble prodrugs of aliphatic or aromatic hindered hydroxyl group containing pharmaceuticals.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to novel water-soluble prodrugs ofaliphatic or aromatic hindered hydroxyl group containingpharmaceuticals. Particularly, the present invention concerns novelwater-soluble phosphonooxymethyl ethers of hindered alcohol and phenolcontaining pharmaceuticals, such as camptothecin, propofol, etoposide,Vitamin E and Cyclosporin A. The present invention also relates tointermediates used to create the final prodrugs as well aspharmaceutical compositions containing the novel compounds.

[0003] 2. Background Art

[0004] The successful delivery of a pharmaceutical to a patient is ofcritical importance in the treatment of disorders. However, the use ofmany clinical drugs with known properties is limited by their very lowwater solubility. As a result of low water solubility these drugs mustbe formulated in co-solvent pharmaceutical vehicles, includingsurfactants. These surfactants have been shown to lead to severe sideeffects in humans that limit the clinical safety of these drugs andtherefore the treatment of several disorders.

[0005] For example, camptothecin is a natural product isolated frombarks of the Chinese camptotheca tree, Camptotheca accuminata. It hasbeen shown to have strong anti-tumor activity in several in vivo animalmodels including major tumor types such as lung, breast, ovary,pancreas, colon and stomach cancer and malignant melanoma. Camptothecininhibits the cellular enzyme DNA topoisomerase I and triggers a cascadeof events leading to apoptosis and programmed cell death. TopoisomeraseI is essential nuclear enzyme responsible for the organization andmodulation of the topological feature of DNA so that a cell mayreplicate, transcribe and repair genetic information.

[0006] The serious drawback of camptothecin is its very limited watersolubility. For biological studies it is necessary to dissolve thecompound in a strong organic solvent (DMSO) or to formulate the drug asa suspension in Tween 80:saline which is an undesirable drug formulationfor human therapy. Recently two analogs of camptothecin with moderatewater solubility have been approved in United States for treatment ofadvanced ovarian cancer (Hycamtin) and colorectal cancer (Camptosar).

[0007] Other drugs, like camptothecin, that have similar problems arecyclosporin A (CsA), propofol, etoposide and Vitamin E(alpha-tocopherol). Like camptothecin, CsA has within its structure asterically hindered alcohol, a secondary alcohol in this case. CsA isformulated in a CremophorEL/ethanol mixture.

[0008] An example of a sterically hindered, poorly water soluble phenolis propofol, an anesthetic.

[0009] Propofol is formulated for i.v. clinical use as a o/w emulsion.Not only is propofol poorly water soluble, but it also causes pain atthe site of injection. This pain must be ameliorated by using lidocaine.Due to the fact that it is formulated as an emulsion, it is difficultand questionable to add other drugs to the formulation and physicalchanges to the formulation such as an increase in oil droplet size canlead to lung embolisms, etc. A water soluble and chemically stableprodrug of propofol would provide several advantages. Such a formulationcould be a simple aqueous solution that could be admixed with otherdrugs. If the prodrug itself was painless, the prodrug may be morepatient friendly, and finally there should be no toxicity due to thevehicle. Other poorly water soluble, sterically hindered phenols are theanticancer drug, etoposide and Vitamin E (alpha-tocopherol).

[0010] The present invention provides a water soluble form of alcoholand phenol containing drugs such as camptothecin and propofol. Withrespect to camptothecin, compounds according to the present inventionsare phosphonooxymethyl ethers of camptothecin in the form of the freeacid and pharmaceutically acceptable salts thereof. The water solubilityof the acid and the salts facilitates preparation of pharmaceuticalformulations. All of the prodrugs according to the present inventionexhibit superior water solubility compared to their respective parentdrugs. The methods developed for the compounds of the present inventioncan be useful for conversion of many other water insoluble medicinalagents having aliphatic or aromatic hindered hydroxyl groups to thewater soluble derivatives.

SUMMARY OF THE INVENTION

[0011] The invention described herein involves new compositions ofmatter. The invention relates to the water soluble phosphonooxymethylderivatives of alcohol and phenol containing pharmaceuticals representedby the general formula I:

[0012] The above formula I is the derivative of ROH, wherein ROHrepresents an alcohol- or phenol-containing drug, such as camptothecin,propofol, etoposide, vitamin E and cyclosporin A. In the above formulaI, n represents an integer of 1 or 2. When n is 2, ROH is preferably aphenol-containing pharmaceutical, such as propofol. Also included aresome drugs for which injectable forms are not possible due to theirinherent poor water solubility. These include danazol,methyltestosterone, iodoquinol and atovaquone. R¹ is hydrogen or analkali metal ion including sodium, potassium or lithium or a protonatedamine or protonated amino acid or any other pharmaceutically acceptablecation. R² is hydrogen or an alkali metal ion including sodium,potassium or lithium or a protonated amine or a protonated amino acid orany other pharmaceutically acceptable cation. After intravenous or oraladministration, the derivatives according to formula I are convertedback to the parent drugs by hydrolysis and/or phosphatase.

[0013] Accordingly, it is an object of the present invention to developderivatives of water insoluble drugs which exhibit good activity andwater solubility.

[0014] It is another object of the present invention to developpharmaceutical compositions of these water soluble compounds, whichcomprises an amount of the compound of formula I and a pharmaceuticallyacceptable carrier.

[0015] It is another object of the present invention to develop drugderivatives having good stability at pH levels suitable for makingpharmaceutical formulations, but quickly break down in vivo underphysiological conditions, to potentially act as prodrugs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings of the present application are explained as follows:

[0017]FIG. 1 illustrates an in vitro enzymatic conversion of propofolprodrug to propofol.

[0018]FIG. 2 illustrates the blood concentration change of propofol withrespect to time from administration of the propofol prodrug or Diprivan®in a dog study.

[0019]FIG. 3 illustrates an in vitro enzymatic conversion ofcamptothecin prodrug to camptothecin.

[0020]FIG. 4 illustrates the correlation between plasma concentration ofcamptothecin from the camptothecin prodrug and from camptothecin inorganic co-solvents for a rat study.

DETAILED DESCRIPTION OF THE INVENTION

[0021] In the present specification, unless otherwise specified or incontext, the following definitions apply.

[0022] “Phosphono-” means the group —P(O)(OH)2 and “phosphonooxymethoxy”or “phosphonooxymethyl ether” means generically the group—OCH₂OP(O)(OH)₂. “Methylthiomethyl” refers to the group —CH₂SCH₃. Thepresent invention also encompasses compounds wherein n=2 such that a“phosphono-di(oxymethyl) ether” generically means the group—OCH₂OCH₂OP(O)(OH)₂.

[0023] “Camptothecin moiety” denotes moiety containing the twenty carboncamptothecin core framework including two nitrogen atoms and four oxygenatoms as represented by the structural formula shown below with theabsolute configuration.

[0024] The numbering system shown above is one used in conventionalcamptothecin derivatives, and is followed throughout the application.For example the notation C20 refers to the carbon atom labeled as “20”.

[0025] “Camptothecin analogue” refers to a compound having the basiccamptothecin core framework. It is to be understood that camptothecinanalogues encompass compounds including but not limited to the followingcompounds: Topotecan, available from SmithKline Beecham, Irinotecan(CPT-11), available from Pharmacia & Upjohn, 9-Aminocamptothecin (9AC),9-Nitrocamptothecin (9NC), GI 147211C, available from Glaxo Wellcome,and DX-8951f (the previous six camptothecin anologues are currentlyunder clinical investigation and are described in a review conducted bythe Pacific West Cancer Fund authored by Claire McDonald (December1997).

[0026] Additionally, several other non-limiting Camptothecin analogueswhich are herein incorporated by reference are disclosed by Sawada etal., Current Pharmaceutical Design, Vol. 1, No. 1, pp 113-132, as wellas U.S. Pat. Nos. 5,646,159, 5,559,235, 5,401,747, 5,364,858, 5,342,947,5,244,903, 5,180,722, 5,122,606, 5,122,526, 5,106,742, 5,053,512,5,049,668, 4,981,968 and 4,894,456.

[0027] Several pharmaceutical compounds including their respectivederivatives of camptothecin contain more than one hydroxyl group, forexample 10-hydroxycamptothecin, topotecan and several others listed inthe above references. It is herein understood that the present inventionmay be applied to more than one hydroxyl group. This may be accomplishedby protecting the additional hydroxyl group prior to derivatization.

[0028] “Phosphono protecting groups” means moieties, which can byemployed to block or protect the phosphono functional group. Preferably,such protecting groups are those that can be removed by methods that donot appreciably affect the rest of the molecule. Suitable phosphonooxyprotecting groups include for example benzyl (denoted by “Bn”), t-butyl,and allyl groups.

[0029] “Pharmaceutically acceptable salt” means a metal or an amine saltof the acidic phosphono group in which the cation does not contributesignificantly to the toxicity or biological activity of the activecompound. Suitable metal salts include lithium, potassium, sodium,calcium, barium, magnesium, zinc, and aluminum salts. Preferred saltsare sodium and potassium salts. Suitable amines salts are for example,ammonia, tromethamine, triethanolamine, ethylenediamine, glucamine,N-methylglucamine, glycine, lysine, ornithine, arginine, ethanolamine,to name but a few. Preferred amine salts are lysine, arginine,N-methylglucamine, and tromethamine salts.

[0030] In the specification and in the claims, the term —OCH₂OP(O)(OH)₂is intended to encompass both the free acid and its pharmaceuticallyacceptable salts, unless the context indicates specifically that thefree acid is intended.

[0031] One aspect of the present invention provides for derivatives ofalcohol and phenol containing pharmaceuticals as shown in formula I:

[0032] The derivatives according to formula I can be prepared accordingto the reaction sequence shown in Scheme 1:

[0033] wherein ROH represents an alcohol- or phenol-containing drug,such as camptothecin, propofol, etoposide, vitamin E, cyclosporin A. Itis to be understood that the above pathway is just one of severalalternate pathways. These alternate pathways will become evident uponreview of the following disclosure and examples.

[0034] An example of the above Scheme 1 can be illustrated using thecompound camptothecin. It is to be understood that these schemes areapplicable to other compounds encompassed by formula I of the presentinvention, such as those listed above. Accordingly, another aspect ofthe present invention provides camptothecin derivatives of according toformula II:

[0035] which include the free acid wherein Z is hydrogen andpharmaceutically acceptable salts thereof wherein Z is metal or amine.Alternatively, formula II includes diacids where Z is metal or amine inboth occurrences.

[0036] The preferred pharmaceutically acceptable salts of a compound offormula II are alkali salts including lithium, sodium, and potassiumsalts; and amine salts including triethylamine, triethanolamine,ethanolamine, arginine, lysine and N-methylglucamine salts.

[0037] The most preferred embodiments of camptothecin derivatives offormula II include the following compounds:(20)-O-phosphonooxymethylcamptothecin,(20)-O-phosphonooxymethylcamptothecin mono- or di-sodium salt,(20)-O-phosphonooxymethylcamptothecin mono or di-potassium salt,(20)-O-phosphonooxymethylcamptothecin mono- or di-arginine salt,(20)-O-phosphonooxymethylcamptothecin mono- or di-lysine salt,(20)-O-phosphonooxymethylcamptothecin mono- or di-N-methylglucamine saltand (20)-O-phosphonooxymethylcamptothecin mono- or di-triethanolaminesalt.

[0038] Compounds of formula II may be prepared directly fromcamptothecin (shown as ©-OH) according to the reaction sequence shown inScheme 2:

[0039] A compound of formula III (methylthiomethyl ether, MTM ether) maybe prepared by treating camptothecin with dimethylsulfoxide/aceticanhydride/acetic acid.

[0040] In the second step shown in Scheme 2, the methylthiomethyl etheris converted to the corresponding protected phosphonooxymethyl ether(compound of formula IV). This is accomplished by treating the MTM etherwith N-iodosuccinamide and protected phosphate HOP(O)(OR)₂. In the thirdstep, the phosphono protecting groups are removed to provide a compoundof formula II. For example, a suitable phosphono protecting group(s) isbenzyl that may be removed by catalytic hydrogenolysis.

[0041] The general process of Scheme 2 for the preparation of a compoundof formula I is more particularly exemplified in Scheme 3:

[0042] In the first step, the free hydroxy group of the camptothecin isconverted to the corresponding methylthiomethyl ether (—OCH₂SCH₃) group.This conversion may be accomplished by reaction with dimethylsulfoxidein the presence of acetic anhydride and acetic acid. This method,commonly known as the Pummer reaction was successfully applied byBristol-Myers Squibb for methylthiomethylation of taxol(Europ.Pat.0604910A1, Bioorg.Med.Chem.Lett.,6,1837,1996). The reactionis usually carried out at room temperature, and for 24-72 hours toproduce the methylthiomethyl ether.

[0043] In the second step of the reaction sequence, the methylthiomethylether is converted to the corresponding protected phosphonooxymethylether. This well-known conversion was successfully applied byBristol-Myers Squibb for phosphonooxymethylation of taxol(Europ.Pat.0604910A1, Bioorg.Med.Chem.Lett.,6,1837,1996). Thus, acompound of formula III is treated with N-iodosuccinamide and protectedphosphoric acid such as dibenzyl phosphate. The reaction is carried outin an inert organic solvent such as tetrahydrofuran and halogenatedhydrocarbon such as methylene chloride and in the presence of molecularsieves. Reaction is carried out at room temperature. N-iodosuccinimideand protected phosphoric acid are used in excess (3-5 equivalents)relative to the methylthiomethyl ether.

[0044] In the third step of the reaction sequence, the phosphonoprotecting groups are removed. The deblocking is accomplished byconventional methods well known in the art such as acid- orbase-catalyzed hydrolysis, hydrogenolysis, reduction, and the like. Forexample, catalytic hydrogenolysis can be used to remove the benzylphosphono-protecting group. Deprotecting methodologies may be found instandard texts, such as T. W. Green and P. G. M. Wutz, Protective groupsin organic sythesis, J. Wiley publishers, New York, N.Y., 1991, pp.47-67.

[0045] The base salts of a compound of formula II may be formed byconventional techniques involving contacting a compound of formula IIfree acid with a metal base or with an amine. Suitable metal basesinclude hydroxides, carbonates and bicarbonates of sodium, potassium,lithium, calcium, barium, magnesium, zinc, and aluminum; and suitableamines include triethylamine, ammonia, lysine, arginine,N-methylglucamine, ethanolamine, procaine, benzathine, dibenzylamine,tromethamine (TRIS), chloroprocaine, choline, diethanolamine,triethanolamine and the like. The base salts may be further purified bychromatography followed by lyophilization or crystallization.

[0046] Compounds of the present invention are phosphonooxymethyl etherpharmaceuticals such as camptothecin, propofol, etoposide, tocopherol,etc. The pharmaceutically acceptable salt forms exhibit improved watersolubility over parent compounds thereby allowing more convenientpharmaceutical formulations. Without being bound by theory, it isbelieved that the phosphonooxymethyl ethers of the present invention areprodrugs of the parent pharmaceuticals; the phosphonooxyethyl moietybeing cleaved upon contact with. phosphatase in vivo to generatesubsequently the parent compound. As shown above, compounds of theinstant invention are effective pharmaceutical or therapeutic agents.

[0047] For example, compounds of formula II of the present invention maybe used in a manner similar to that of camptothecin. The structure ofthe camptothecin prodrug is shown above. Therefore, an oncologistskilled in the art of cancer treatment will be able to ascertain,without undue experimentation, an appropriate treatment protocol foradministering a compound of the present invention. The dosage, mode andschedule of administration of compounds of this invention are notparticularly restricted, and will vary with the particular compoundemployed. Thus a compound of the formula II may be administrated via anysuitable route of administration, preferable parenterally; the dosagemay be, for example, in the range of about 0.1 to about 100 mg/kg ofbody weight, or about 5 to 500 mg/m2. Compounds of formula II may alsobe administrated orally; oral dosage may be in the range of about 5 toabout 500 mg/kg of body weight. The actual dose used will vary accordingto the particular composition of formulated, the route ofadministration, and the particular site, host and type of tumor beingtreated. Many factors that modify the action of the drug will be takeninto account in determining the dosage including age, sex, diet and thephysical conditions of the patient.

[0048] Another example is the propofol prodrug according to formula I ofthe present invention. The structure of the propofol prodrug is shownbelow:

[0049] In the above formula for the propofol prodrug, Z is the same asdefined above for formula II. Therefore, an anesthesiologist skilled inthe art of anesthesia will be able to ascertain, without undueexperimentation, an appropriate treatment protocol for administering acompound of the present invention. The dosage, mode and schedule ofadministration of compounds of this invention are not particularlyrestricted, and will vary with the particular compound employed. Thus, acompound of formula I such as the propofol prodrug may be administeredvia any suitable route of administration, preferably parenterally; thedosage may be, for example, in the range of 0.5 to 10 mg/kg administeredaccording to procedures for induction of general anesthesia ormaintenance of general anesthesia. Alternatively, the compound offormula I may be administered by parenteral infusion, the dosage may be,for example, in the range of 2 μg/kg/min to 800 μg/kg/min administeredaccording to procedures for maintenance of general anesthesia,initiation and maintenance of MAC sedation or initiation and maintenanceof ICU sedation.

[0050] The present invention also provides pharmaceutical compositionscontaining a pharmaceutically effective amount of compound of formula Iin combination with one or more pharmaceutically acceptable carriers,excipients, diluents or adjuvants. For example, compounds of the presentinvention may be formulated in the form of tablets, pills, powdermixtures, capsules, injectables, solutions, suppositories, emulsions,dispersions, food premix, and in other suitable forms. They may also bemanufactured in the form of sterile solid compositions, for example,freeze-dried and, if desired, combined with other pharmaceuticallyacceptable excipients. Such solid compositions can be reconstituted withsterile water, physiological saline, or a mixture of water and anorganic solvent, such as propylene glycol, ethanol, and the like, orsome other sterile injectable medium immediately before use ofparenteral administration.

[0051] Typical of pharmaceutically acceptable carriers are, for example,manitol, urea, dextrans, lactose, non-reducing sugars, potato and maizestarches, magnesium stearate, talc, vegetable oils, polyalkyleneglycols, ethyl cellulose, poly(vinyl-pyrrolidone), calcium carbonate,ethyloleate, isopropyl myristate, benzyl benzoate, sodium carbonate,gelatin, potassium carbonate, silicic acid. The pharmaceuticalpreparation may also contain non toxic auxiliary substances such asemulsifying, preserving, wetting agents, and the like as for example,sorbitan monolaurate, triethanolamine oleate, polyoxyethylenemonostearate, glyceryl tripalmitate, dioctyl sodium sulfosuccinate, andthe like.

[0052] In the following experimental procedures, all temperatures areunderstood to be in Centigrade (C) when not specified. The nuclearmagnetic resonance (NMR) spectral characteristics refer to chemicalshifts (δ) expressed in parts per million (ppm) versus tetramethylsilane(TMS) as reference standard. The relative area reported for the variousshifts in the proton NMR spectral data corresponds to the number ofhydrogen atoms of a particular functional type in the molecule. Thenature of the shifts as to multiplicity is reported-as broad singlet(bs), broad doublet (bd), broad triplet (bt), broad quartet (bq),singlet (s), multiplet (m), doublet (d), quartet (q), triplet (t),doublet of doublet (dd), doublet of triplet (dt), and doublet of quartet(dq). The solvents employed for taking NMR spectra are acetone-d6(deuterated acetone) DMSO-d6 (perdeuterodimethylsulfoxide), D₂O(deuterated water), CDCl3 (deuterochloroform) and other conventionaldeuterated solvents.

[0053] The abbreviations used herein are conventional abbreviationswidely employed in the art. Some of which are: MS (mass spectrometry);HRMS (high resolution mass spectrometry); Ac (acetyl); Ph (phenyl); FAB(fast atom bombardment) min (minute); h or hrs (hour(s)); NIS(N-iodosuccinimide); DMSO (dimethylsulfoxide); THF (tetrahydrofuran).

[0054] The following examples are provided to illustrate the synthesisof representative compounds of the instant invention and are not to beconstrued as limiting the scope of the invention in any manner. Oneskilled in the art will be able to adapt these methods, without undueexperimentation, to the synthesis of compounds within the scope of thisinvention but not specifically disclosed. For example, in the followingexamples, specific salts are employed, however, these salts are not tobe construed as limiting. An example of this situation is the repeateduse of a silver salt of dibenzylphosphate. Tetralkyl ammonium salts,such as tetramethyl ammonium salts or other alkali metal salts may beused in lieu of the silver salt.

EXAMPLES

[0055] I. Synthesis of O-Phosphonooxymethylpropofol

[0056] Ia. Synthesis of O-methylthiomethylpropofol:

[0057] To a stirred suspension of sodium hydride (150 mg, 6.2 mmol) indry HMPA (10 mL), kept under an argon atmosphere, was added dropwisepropofol (1.1 mL of 97%, 5.7 mmol) over 15 minutes. The reaction mixturewas then stirred at room temperature for an additional 30 minutes. Tothis mixture was added dropwise chloromethyl methyl sulfide (550 μl of95%, 6.2 mmol) and then stirred at room temperature. After 20 hours, thereaction mixture was partitioned with stirring between water (10 mL) andbenzene (20 mL). The aqueous layer was separated and extracted withbenzene (10 mL). The benzene fractions were combined, washed with water(2×3 mL), dried over sodium sulfate, and evaporated under reducedpressure. The resulting oily residue was subjected to columnchromatography (silica gel, hexane, then 4:1 hexane/chloroform) to give1.15 g (85% yield) of the title compound as a colorless oil.

[0058] EIMS: [M+], m/z 238.

[0059]¹H NMR (300 MHz, CDCl₃, δ): 1.24 (d, J=6.9 Hz, 12H), 2.37 (s, 3H),3.37 (hept, J=6.9 Hz, 2H), 4.86 (s, 2H), 7.12 (s, 3H). ¹³C NMR (75 MHz,CDCl₃, δ): 15.40, 23.98, 26.68, 78.12, 124.04, 125.05, 141.74, 152.20.

[0060] Ib. Synthesis of O-chloromethylpropofol:

[0061] To a stirred solution of O-methylthiomethylpropofol (3.00 g, 12.5mmol) in dry methylene chloride (30 mL), kept under an argon atmosphere,was added a 1M solution of SO₂Cl₂ in dry methylene chloride (12.2 mL,12.2 mmol) at 5° C. over five minutes. The reaction mixture was stirredfor 10 minutes at the same temperature and then for three hours at roomtemperature. The solvent was evaporated under reduced pressure and thebrown residual oil was purified by flash column chromatography (silicagel, 1:20 hexane/ethyl acetate) to give 2.36 g (83% yield) of the titlecompound as a yellow oil.

[0062] CIMS (NH₃): [M]⁺, m/z 226, [MH+NH₃]⁺, m/z 244.

[0063]¹H NMR (300 MHz, CDCl₃, δ): 1.22 (d, J=6.9 Hz, 12H), 3.35 (hept,J=6.9 Hz, 2H), 5.76 (s, 2H), 7.15 (m, 3H). ¹³C NMR (75 MHz, CDCl₃,δ)23.93, 26.84, 83.34, 124.34, 125.95, 141.34, 150.93.

[0064] Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester(Route-1):

[0065] A mixture of O-chloromethylpropofol (2.20 g, 9.7 mmol), silverdibenzylphosphate (3.85 g, 10.0 mmol) and dry toluene (50 mL) wasrefluxed under an argon atmosphere for 45 minutes. The mixture wascooled down to room temperature and filtered. After the solvent wasevaporated in vacuo, the oily residue was purified by silica gel flashcolumn chromatography (9:1 hexane/ethyl acetate and then 1:1hexane/ethyl acetate) to give 4.43 g (98% yield) of the title compoundas a yellow oil.

[0066] CIMS (NH₃): [MH]⁺, m/z 469, [MH+NH3]⁺, m/z 486.

[0067]¹H NMR (300 MHz, CDCl₃, δ): 1.17 (d, J=6.8 Hz, 12H), 3.33 (hept,J=6.9 Hz, 2H), 5.00 (d, J=7.8 Hz, 2H), 5.01 (d, J=7.8 Hz, 2H), 5.42 (d,J=9.9 Hz, 2H), 7.12 (m, 3H), 7.32 (m, 10H). ¹³C NMR (75 MHz, CDCl₃, δ):23.79, 26.57, 69.15, 69.23, 94.14, 94.20, 124.07, 125.62, 127.70,128.44, 135.42, 135.51, 141.50, 151.07.

[0068] Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester(Alternate Route-1):

[0069] To a stirred solution of O-methylthiomethylpropofol (1.45 g, 6.08mmol) in dry methylene chloride (15 mL) under an argon atmosphere at0-5° C. was added a 1M solution of SO₂Cl₂ in dry methylene chloride (6.5mL, 6.5 mmol) over five minutes. The reaction mixture was stirred for 10minutes at 5° C. and three hours at room temperature. Then the solventwas evaporated under reduced pressure. The residual oil was dissolved intoluene (ACS-grade, 20 mL); silver dibenzylphosphate (3.50 g, 9.1 mmol)was added, and the resulting mixture was refluxed for 45 minutes. Thebrown reaction mixture was cooled down to room temperature and filtered.After the solvent was evaporated in vacuo, the oily residue was purifiedby silica gel flash column chromatography (9:1 hexane/ethyl acetate,then 1:1 hexane/ethyl acetate) to give 2.41 g (85% yield) of the titlecompound as a yellow oil. This product had the same Rf (TLC) and ¹H NMRspectrum (300 MHz, CDCl₃) as an authentic sample.

[0070] Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester(Alternate Route-2):

[0071] To a stirred suspension of sodium hydride (41 mg of 60%dispersion in mineral oil, 1.02 mmol) in dry dimethoxyethane (1.5 mL)under an argon atmosphere was added dropwise propofol (200 μl of 97%,1.04 mmol) over 5 minutes and the resulting mixture was stirred for anadditional 15 minutes. The resulting homogeneous solution was addeddropwise to a stirred solution of chloroiodomethane (4.0 mL, 53 mmol) indry dimethoxyethane (4 mL) over 15 minutes. This reaction mixture wasstirred for two hours, filtered, and then the solvent and the excess ofchloroiodomethane were evaporated. The residual oil was dissolved intoluene (HPLC-grade, 10 mL). To this solution was added silverdibenzylphosphate (400 mg, 1.04 mmol), and the resulting mixture wasrefluxed for 10 minutes. After the reaction mixture was cooled down toroom temperature and filtered, the solvent was evaporated in vacuo. Theoily residue was purified by silica gel flash column chromatography (9:1hexane/ethyl acetate and then 1:1 hexane/ethyl acetate) to give 205 mg(42% yield) of the title compound as a yellow oil. This product had thesame Rf (TLC) and ¹H NMR spectrum (300 MHz, CDCl₃) as an authenticsample.

[0072] Further to the above reaction Ic (alternate route-2) it isunderstood that other reagents may be used depending on the desiredcompound. For-example, when, a compound of formula I wherein n=2 isdesired, the chloroiodomethane may be substituted with a compound suchas X—CH2-O—CH2-Cl, wherein X is a good leaving group.

[0073] Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester(Alternate Route-3):

[0074] To a stirred solution of O-methylthiomethylpropofol (91 mg, 0.38mmol) in dry methylene chloride (2 mL) under an argon atmosphere wereadded powdered, activated 4A molecular sieves (100 mg), and then asolution of dibenzylphosphate (127 mg, 0.45 mmol) and N-iodosuccinimide(102 mg of 95%, 0.43 mmol) in tetrahydrofuran (2 mL). The reactionmixture was stirred at room temperature for one hour, filtered, anddiluted with methylene chloride (30 mL). The resulting solution waswashed with a solution of sodium thiosulfate (2 mL of a 1M solution), asaturated solution of sodium bicarbonate (3 mL), brine (5 mL), driedover a mixture of sodium sulfate and magnesium sulfate, filtered, andconcentrated in vacuo. The oily residue was purified by silica gel flashcolumn chromatography (1:1 hexane/ethyl acetate) to give 120 mg (67%yield) of the title compound as a yellow oil. This product had the sameRf (TLC) and ¹H NMR spectrum (300 MHz, CDCl₃) as an authentic sample.

[0075] Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl

[0076] ester (Alternate Route-4):

[0077] To a solution of propofol (38 mg of 97%, 0.21 mmol) in methylenechloride (1 mL) was added tetrabutylammonium bromide (10 mg, 0.03 mmol)and a solution of sodium hydroxide (40 mg, 1 mmol) in water (0.2 mL).The heterogeneous mixture was stirred for 15 minutes. Then a solution ofchloromethyl dibenzylphosphate (104 mg, 0.32 mmol) in methylene chloride(1 mL) was added and the reaction mixture was stirred vigorously foreight hours. The mixture was then diluted with methylene chloride (10mL), washed with water (2 mL), dried over sodium sulfate, filtered, andevaporated in vacuo. The oily residue was purified by silica gel flashcolumn chromatography (hexane, 20:1 hexane/ethyl acetate, and 10:1hexane/ethyl acetate) to give 44 mg (45% yield) of the title compound asa yellow oil. This product had the same Rf (TLC) and ¹H NMR spectrum(300 MHz, CDCl₃) as an authentic sample.

[0078] Further to the above reaction Ic (alternate route-4) it is to beunderstood that the reagent:

[0079] can be generically represented by the following formula:

[0080] wherein X represents a leaving group, R3 and R4 are each ahydrogen atom, an organic group or an inorganic group and Y is aphosphate protecting group. Examples of leaving groups include chlorine,bromine, iodine, tosylate or any other suitable leaving group. Examplesof phosphate protecting groups include protecting groups thattemporarily block the reactivity of the phosphate group and permitselective displacement with the nucleophilic displacement reaction.Examples of such blocking groups include but are not limited to benzyl,allyl, tertiary butyl and isopropyl, ethyl and β-cyanoethyl.

[0081] Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester(Alternate Route-5):

[0082] To a stirred suspension of sodium hydride (36 mg of a 60%dispersion in mineral oil, 0.91 mmol) in dry dimethoxyethane (2 mL)under an argon atmosphere was added dropwise propofol (172 μl of 97%,0.90 mmol) over five minutes. The resulting mixture was stirred at roomtemperature for an additional 20 minutes. To the mixture was then addedthe solution of formaldehyde bis-(dibenzylphosphono)acetal (500 mg, 0.88mmol) in dry dimethoxyethane (3 mL). The reaction mixture was stirred atroom temperature for 20 hours and then at 70° C. for 2.5 hours. Themixture was then filtered and the solvent was evaporated in vacuo. Theoily residue was purified by silica gel flash column chromatography(hexane, 10:1 hexane/ethyl acetate, and then 1:1 hexane/ethyl acetate)to give 29 mg (7% yield) of the title compound as a yellow oil. Thisproduct had the same Rf (TLC) and ¹H NMR spectrum (300 MHz, CDCl₃) as anauthentic sample.

[0083] Id. Synthesis of O-phosphonooxymethylpropofol:

[0084] To a solution of O-phosphonooxymethylpropofol dibenzyl ester (115mg, 0.245 mmol) in methanol (10 mL) was added palladium on carbon (10%,20 mg). This mixture was stirred under an atmosphere of hydrogen (1 atm)for 1.5 hour. The catalyst was removed by filtration through Celite, andthe filtrate was evaporated at reduced pressure to give 70.5 mg (100%yield) of the title compound as a colorless oil, unstable on standing atroom temperature.

[0085] FABMS-(GLY) : [M−H]⁻, m/z 287.

[0086]¹H NMR (300 MHz, acetone-d₆, δ): 1.19 (d, J=6.8 Hz, 12H), 3.46(sext, J=6.8 Hz, 2H), 5.45 (d, J=9.7 Hz, 2H), 7.15 (m, 3H). ¹³C NMR (75MHz, acetone-d₆, δ): 24.2178, 27.1496, 94.63, 94.65, 124.08, 126.30,142.46, 152.32.

[0087] Ie. Synthesis of O-phosphonooxymethylpropofol disodium salt:

[0088] To a solution of O-phosphonooxymethylpropofol dibenzyl ester(1.05 g, 2.24 mmol) in tetrahydrofuran (100 mL) was added water (5 mL)and palladium on carbon (10%, 300 mg). This mixture was stirred underhydrogen (1 atm) for one hour. The catalyst was removed by filtrationthrough Celite, and the filtrate was treated with a solution of sodiumcarbonate hydrate (263 mg in 3 mL of water, 2.12 mmol). THF wasevaporated under reduced pressure and the residual water solution wasextracted with ether (3×3 mL). The aqueous layer was evaporated todryness (argon stream or rotary evaporator) and the resulting solid wasdried overnight in vacuo, washed with ether (4×4 mL), hexane (2×4 mL),and again dried in vacuo to provide 655 mg (93% yield) of the titlecompound as a white powder.

[0089] FABMS-(GLY): [M−2Na+H]—, m/z 287.

[0090]¹H NMR (300 MHz, D₂O, δ): 1.22 (d, J=7.0 Hz, 12H), 3.46 (hept,J=6.9 Hz, 2H), 5.27 (d, J=7.5 Hz, 2H), 7.28 (m, 3H).

[0091] II. Synthesis of O-Phosphonooxymethyl-alpha-tocopherol

[0092] IIa. Synthesis of O-phosphonooxymethyl-alpha-tocopherol dibenzylester:

[0093] To a solution of chloromethyl dibenzylphosphate (323 mg, 0.98mmol), alphatocopherol (409 mg of 97%, 0.92 mmol), andtetrabutylammonium bromide (301 mg, 0.92 mmol) in benzene (5 mL) wasadded an aqueous solution of sodium hydroxide (150 mg in 0.2 mL ofwater, 3.7 mmol). The resulting reaction mixture was vigorously stirredat room temperature for two hours under an argon atmosphere. The mixturewas then diluted with benzene (10 mL), washed with water (3×3 mL), driedover magnesium sulfate, filtered, and evaporated under reduced pressure.The brown oily residue was purified by silica gel flash columnchromatography (10:1 hexane/ethyl acetate) to give 336 mg (51% yield) ofthe title compound as a yellow oil.

[0094] FABMS+(NBA): [M]⁺, m/z 720.

[0095]¹H NMR (500 MHz, CDCl₃, δ): 0.85 (m, 12H), 1.21 (s, 3H), 1.27 (m,24H), 1.75 (m, 2H), 2.06 (s, 3H), 2.11 (s, 3H), 2.14 (s, 3H), 2.54 (t,J=6.8 Hz, 2H), 4.97 (m, 4H), 5.20 (d, J=9.3 Hz, 2H), 7.31 (m, 10H).

[0096] IIb. Synthesis of O-phosphonooxymethyl-alpha-tocopherol:

[0097] To a solution of O-phosphonooxymethyl-alpha-tocopherol dibenzylester (88 mg, 0.12 mmol) in tetrahydrofuran (10 mL) was added palladiumon carbon (10%, 15 mg). The mixture was stirred under an atmosphere ofhydrogen (1 atm) for 10 minutes (the reaction was complete after 5minutes as judged by TLC). The catalyst was removed by filtrationthrough Celite, the filtrate was evaporated at reduced pressure, andthen dried in vacuo. The title compound was obtained in an amount of 70mg (100% yield) as a brownish oil, which was unstable at roomtemperature.

[0098] FABMS+(NBA) : [M]⁺, m/z 540, [M +Na]⁺, m/z 563; (NBA+Li) [M+Li]⁺, m/z 547

[0099] IIc. Synthesis of O-phosphonooxymethyl-alpha-tocopherol disodiumsalt:

[0100] To a solution of O-phosphonooxymethyl-alpha-tocopherol dibenzylester (100 mg, 0.14 mmol) in tetrahydrofuran (10 mL) was added palladiumon carbon (10%, 18 mg) . The mixture was stirred under an atmosphere ofhydrogen (1 atm) for 5 minutes. The catalyst was removed by filtrationthrough Celite, the filtrate was evaporated at room temperature atreduced pressure, and the resulting residue was dissolved in ether (2mL). The ether solution was then treated with an aqueous solution ofsodium hydroxide (11.2 mg in 100 mL of water, 0.28 mmol), and theresulting mixture was stirred at room temperature for 10 min. The etherphase was removed and the aqueous phase was washed with ether (3×3 mL)and then dried in vacuo for 20 hours to give 73 mg (89% yield) of thetitle compound as a gray solid.

[0101] FABMS+(TG/G): [MH]⁺, m/z 585, [M+Na]⁺, m/z 607

[0102] The synthesis of water soluble derivatives of camptothecin willalso be further detailed as follows:

[0103] III. Synthesis of 20-O-Phosphonooxymethylcamptothecin

[0104] IIIa. Synthesis of 20-O-methylthiomethylcamptothecin:

[0105] To a suspension of camptothecin (5.0 g, 14.3 mmol) indimethylsulfoxide (250 mL) was added acetic anhydride (125 mL) andacetic acid (35 mL). The heterogeneous mixture was vigorously stirred atroom temperature for 24 hours, poured into ice (800 mL), stirred for 30minutes, and then extracted with methylene chloride (4×100 mL). Thecombined methylene chloride extracts were washed with water (2×100 mL)and dried over magnesium sulfate. The methylene chloride was removed atreduced pressure to give a brownish solid. The solid was dissolved in aminimum volume of methylene chloride. This solution was filtered anddiluted with a 10-fold excess of hexane and then kept overnight in therefrigerator. The precipitated solid was filtered off, washed severaltimes with hexane, and dried to give 5.38 g (92% yield) of the titlecompound as a light brown powder. α^(D) ₂₀-123.6° (c 0.55, CHCl₃).

[0106] FABMS+(NBA): [MH]⁺, m/z 409.

[0107]¹H NMR (400 MHz, CDCl₃, δ): 0.93 (t, J=7.2 Hz, 3H), 2.11 (sext,J=7.6 Hz, 1H), 2.29 (sext, J=7.6 Hz, 1H), 2.30 (s, 3H), 4.58 (s, 2H),5.33 (s, 2H), 5.40 (d, J=17.2 Hz, 1H), 5.62 (d, J=17.3 Hz, 1H), 7.48 (s,1H), 7.69 (t, J=7.1 Hz, 1H), 7.86 (t, J=7.1 Hz, 1H), 7.96 (d, J=8.1 Hz,1H), 8.25 (d, J=8.5 Hz, 1H), 8.42 (s, 1H).

[0108]¹³C NMR (75 MHz, CDCl₃, δ): 7.76, 14.89, 33.90, 49.92, 66.68,71.02, 76.57, 97.51, 122.63, 128.02, 128.09, 128.30, 129.71, 130.64,131.11, 145.14, 146.10, 148.88, 152.27, 157.43, 169.34, 169.73.

[0109] IIIb. Synthesis of 20-O-phosphonooxymethylcamptothecin dibenzylester:

[0110] To a well stirred suspension of 20-O-methylthiomethylcamptothecin(1.00 g, 2.44 mmol) and powdered, activated 4 Å molecular sieves (5 g)in tetrahydrofuran (20 mL) was added a suspension of N-iodosuccinimide(2.00 g of 95%, 8.44 mmol) and dibenzylphosphate (2.20 g, 7.83 mmol) inmethylene chloride (12 mL). The resulting mixture was vigorously stirredat room temperature for 30 minutes, filtered, and diluted with ethylacetate (300 mL). The solution was washed with aqueous sodiumthiosulfate (10%, 2×15 mL), water (2×20 mL), brine (50 mL), and driedover magnesium sulfate. The mixture was filtered and the solvent wasevaporated under reduced pressure. The brown oily residue was purifiedby silica gel flash column chromatography (98:2 ethyl acetate/methanol)and dried in vacuo overnight to give 1.19 g (76% yield) of the titlecompound as a yellow foam. α^(D) ₂₀-43.1° (c 0.55, CHCl₃).

[0111] FABMS+(NBA): [MH]⁺, m/z 639.

[0112]¹H NMR (400 MHz, CDCl₃, δ): 0.91 (t, J=7.4 Hz, 3H), 2.09 (sext,J=7.4 Hz, 1H), 2.26 (sext, J=7.4 Hz, 1H), 5.06 (m, 4H), 5.28 (m, 3H),5.35 (d, J=17.0 Hz, 1H), 5.48 (2xd, J=10.5 Hz, 1H), 5.64 (d, J=17.3 Hz,1H), 7.59 (s, 1H), 7.67 (t, J=7.0 Hz, 1H), 7.80 (t, J=7.1 Hz, 1H), 7.94(d, J=8.0 Hz, 1H), 8.13 (d, =8.5 Hz, 1H), 8.35 (s, 1H).

[0113]¹³C NMR (100 MHz, CDCl₃, δ): 7.73, 29.53, 32.49, 49.86, 66.74,69.37, 69.44, 78.48, 88.99, 89.04, 98.09, 121.55, 127.65, 127.70,127.90, 128.01, 128.25, 128.35, 128.36, 129.62, 130.48, 130.97, 135.45,135.55, 145.47, 145.82, 148.76, 152.15, 157.18, 168.67.

[0114] IIIc. Synthesis of 20-O-phosphonooxymethylcamptothecin:

[0115] To a solution of 20-O-phosphonooxymethylcamptothecin dibenzylester (500 mg, 0.78 mmol) in tetrahydrofuran (100 mL) and water (5 mL)was added palladium on carbon (10%, 500 mg). This mixture was stirredunder an atmosphere of hydrogen (1 atm) for 35 minutes. The catalyst wasremoved by filtration through Celite. The Celite was then washed withtetrahydrofuran (300 mL) and the combined filtrates were evaporated atreduced pressure. The resulting green solid was washed with ether (2×20mL), hexane (50 mL), dried in vacuo, and then dissolved in hot methanol(60 mL). The solution was filtered, concentrated at reduced pressure to˜10 mL volume. After standing at room temperature for one hour, thesolution was placed in the refrigerator overnight. The crystallineprecipitate that had formed overnight was filtered off and dried invacuo to give 155 mg of the title compound as a yellow solid. Thefiltrate was concentrated to ˜1 mL volume and kept in the refrigeratorfor one hour to give an additional 28 mg of the product. Total yield:183 mg (51%).

[0116] FABMS+(NBA) : [MH]⁺, m/z 459, [M +Na]⁺, m/z 481.

[0117]¹H NMR (400 MHz, D₂O, δ): 0.95 (t, J=7.5 Hz, 3H), 2.25 (m, 2H),4.98 (d, J=5.0 Hz, 2H), 5.14 (2xd, J=9.3 Hz, 1H), 5.22 (2xd, J=8.9 Hz,1H), 5.48 (d, J=17.0 Hz, 1H), 5.60 (d, J=16.9 Hz, 1H), 7.54 (s, 1H),7.56 (t, J=7.7 Hz, 1H), 7.77 (t, J=7.2 Hz, 1H), 7.86 (d, J=8.2 Hz, 1H),8.01 (d, J=8.5 Hz, 1H), 8.44 (s, 1H).

[0118] Chemical structure and purity of the product were also confirmedby ¹H NMR spectroscopy of its disodium salt, formed from the acid andtwo mole equivalents of sodium bicarbonate in D₂O.

[0119] IIIc. Synthesis of 20-O-phosphonooxymethylcamptothecin (AlternateRun):

[0120] To a solution of 20-O-phosphonooxymethylcamptothecin dibenzylester (500 mg, 0.78 mmol) in tetrahydrofuran (100 mL) and water (5 mL)was added palladium on carbon (10%, 500 mg). The mixture was stirredunder an atmosphere of hydrogen (1 atm) for 30 minutes. The catalyst wasremoved by filtration through Celite. Celite was washed withtetrahydrofuran (2×100 mL), and the combined filtrates were treated withan aqueous solution of sodium carbonate hydrate (97 mg in 2 mL water,0.78 mmol). THF was evaporated at reduced pressure and the heterogeneousaqueous residue was diluted with water (10 mL) and extracted with ethylacetate (2×3 mL). The resulting yellow homogeneous solution wasacidified with hydrochloric acid (10%) to pH=1. The resultingprecipitate was filtered off and dried in vacuo overnight to give 145 mg(41% yield) of the title compound as a yellow solid.

[0121] IIId. Synthesis of 20-O-phosphonooxymethylcamptothecin disodiumsalt:

[0122] To a suspension of 20-O-phosphonooxymethylcampto-thecin (5 mg,10.9 μmol) in deuterium oxide (0.5 mL) was added a deuterium oxidesolution of sodium bicarbonate (50 μl of 0.44 M solution=22 μmol). Theheterogeneous mixture was sonicated for a few minutes to give a yellowhomogenous solution of the title product. ¹H NMR (400 MHz, D₂O, after 10min., 96% lactone, 4% carboxylate, δ): 1.05 (t, J=7.2 Hz, 3H), 2.27 (m,2H), 4.57 (d, J=18.8 Hz, 1H), 4.70 (d, J=18.9 Hz, 1H), 5.06 (dd, J=8.3,J=5.4 Hz, 1H), 5.18 (dd, J=7.6, J=5.5 Hz, 1H), 5.45 (d, J=16.7 Hz, 1H),5.59 (d, J=16.8 Hz, 1H), 7.34 (t, J=7.1 Hz, 1H), 7.41 (s, 1H), 7.60 (m,2H), 7.81 (d, J=8.3 Hz, 1H), 8.17 (s, 1H).

[0123] IIId. Synthesis of 20-O-phosphonooxymethylcamptothecin disodiumsalt (Alternate Run 1):

[0124] To a solution of 20-O-phosphonooxymethylcamptothecin dibenzylester (78 mg, 0.122 mmol) in tetrahydrofuran (10 mL) and water (3 mL)was, added palladium on carbon (10%, 80 mg). The mixture was stirredunder an atmosphere of hydrogen (1 atm) for 30 minutes. The catalyst wasremoved by filtration through Celite and the filtrate was treated withan aqueous solution of sodium bicarbonate (20 mg in 0.5 mL of water,0.238 mmol). The yellow precipitate was filtered off, washed withmethylene chloride, and dried in vacuo to give 35 mg (57% yield) of thetitle compound (light brown solid) as a mixture of its lactone form(82%) and its carboxylate form (18%) (by ¹H NMR).

[0125] IIId. Synthesis of 20-O-phosphonooxymethylcamptothecin disodiumsalt (Alternate Run 2):

[0126] To a solution of 20-O-phosphonooxymethylcamptothecin dibenzylester (500 mg, 0.78 mmol) in tetrahydrofuran (100 mL) and water (5 mL)was added palladium on carbon (10%, 500 mg). This mixture was stirredunder an atmosphere of hydrogen (1 atm) for 30 minutes. The catalyst wasremoved by filtration through Celite. Celite was washed withtetrahydrofuran (50 mL), and the combined filtrates were treated with anaqueous solution of sodium carbonate hydrate (90 mg in 2 mL of water,0.72 mmol). Tetrahydrofuran was evaporated at reduced pressure, and theresidue was dissolved in water (15 mL). The heterogeneous mixture wasextracted with ethyl acetate (2×15 mL) and ether (20 mL) and theresulting aqueous homogenous solution was evaporated to dryness under astream of argon at room temperature. The residue was dried in vacuoovernight to give 290 mg (80% yield) of the title compound (orangesolid) as a mixture of its lactone form (60%), its carboxylate form(40%), and a small amount of byproducts (by ¹H NMR).

[0127] IIIe. Synthesis of 20-O-phosphonooxymethylcamptothecin monosodiumsalt:

[0128] To a continuously sonicated suspension of20-O-phosphonooxymethylcamptothecin (5 mg, 10 μmol) in deuterium oxide(0.5 mL) was added dropwise a deuterium oxide solution of sodiumbicarbonate until complete homogenization was achieved (21 μl of 0.44 Msolution=9.2 μmol). A yellow homogenous solution of the title compoundwas obtained.

[0129]¹H NMR (400 MHz, D₂O, δ): 1.00 (t, J=7.2 Hz, 3H), 2.23 (m, 2H),4.40 (d, J=18.8 Hz, 1H), 4.50 (d, J=18.8 Hz, 1H), 5.10 (dd, J=9.7, J=5.9Hz, 1H), 5.26 (dd, J=9.0, J=6.1 Hz, 1H), 5.39 (d, J=16.7 Hz, 1H), 5.50(d, J=16.7 Hz, 1H), 7.20 (t, J=7.3 Hz, 1H), 7.28 (s, 1H), 7.46 (m, 2H),7.66 (d, J=8.4 Hz, 1H), 8.02 (s, 1H).

[0130] IIIf. Synthesis of 20-O-phosphonooxymethylcamptothecin lysinesalt:

[0131] To a continuously sonicated suspension of20-O-phosphonooxymethylcamptothecin (5 mg, 10 μmol) in deuterium oxide(0.5 mL) was added dropwise a deuterium oxide solution of L-lysine (25μl of 0.43 M solution=10.7 μmol) until complete homogenization wasachieved. A yellow homogenous solution of the title compound wasobtained.

[0132]¹H NMR (400 MHz, D₂O, 94% lactone, 6% carboxylate, δ): 1.02 (t,J=7.2 Hz, 1H), 1.49 (m, 2H), 1.73 (m, 2H), 1.88 (m, 2H), 2.25 (m, 2H),3.03 (t, J=7.5 Hz, 2H), 3.76 (t, J=6.0 Hz, 1H), 4.43 (d, J=19.0 Hz, 1H),4.52 (d, J=18.9 Hz, 1H), 5.11 (dd, J=9.7, J=5.8 Hz, 1H), 5.27 (dd,J=9.2, J=5.8 Hz, 1H), 5.41 (d, J=16.7 Hz, 1H), 5.53 (d, J=16.7 Hz, 1H),7.23 (t, J=7.4 Hz, 1H), 7.30 (s, 1H), 7.49 (m, 2H), 7.68 (d, J=8.4 Hz,1H), 7.04 (s, 1H)

[0133] IIIg. Synthesis of 20-O-phosphonooxymethylcamptothecin argininesalt:

[0134] To a continuously sonicated suspension of20-O-phosphonooxymethylcamptothecin (5 mg, 10 μmol) in deuterium oxide(0.5 mL) was added dropwise a deuterium oxide solution of L-arginine (27μl of 0.40 M, 10.8 μmol) until complete homogenization was achieved. Ayellow homogenous solution of the title compound was obtained.

[0135]¹H NMR (400 MHz, D₂O, δ): 1.02 (t, J=7.1 Hz, 1H), 1.66 (m. 2),1.89 (m, 2H), 2.25 (m, 2H), 3.20 (t, J=6.8 Hz, 2H), 3.77 (t, J=6.0 Hz,1H), 4.40 (d, J=19.0 Hz, 1H), 4.49 (d, J=18.8 Hz, 1H), 5.12 (dd, J=9.7,J=6.0 Hz, 1H), 5.29 (dd, J=8.8, J=6.1 Hz, 1H), 5.40 (d, J=16.7 Hz, 1H),5.51 (d, J=16, 7 Hz, 1H), 7.20 (t, J=7.3 Hz, 1H), 7.29 (s, 1H), 7.47 (m,2H), 7.66 (d, J=8.3 Hz, 1H), 8.03 (s, 1H).

[0136] IIIh. Synthesis of 20-O-phosphonooxymethylcamptothecinN-methylglucamine salt:

[0137] To a continuously sonicated suspension of20-O-phosphonooxymethylcamptothecin (5 mg, 10.9 μmol) in deuterium oxide(0.5 mL) was added dropwise a deuterium oxide solution of(D)-N-methylglucamine (21 μl of 0.51 M solution=10.7 μmol) untilcomplete homogenization was achieved. A yellow homogenous solution ofthe title compound was obtained.

[0138]¹H NMR (400 MHz, D₂O, δ): 1.02 (t, J=7.3 Hz, 3H), 2.25 (m, 2H),2.78 (s, 3H), 3.20 (m, 2H), 3.65 (m, 2H), 3.80 (m, 3H), 4.11 (m, 1H),4.44 (d, J=18.9 Hz, 1H), 4.53 (d, J=19.0 Hz, 1H), 5.12 (dd, J=9.8, J=5.9Hz, 1H), 5.27 (dd, J=9.2, J=5.9 Hz, 1H), 5.41 (d, J=16.7 Hz, 1H), 5.53(d, J=16.7 Hz, 1H), 7.23 (t, J=7.4 Hz, 1H), 7.49 (m, 2H), 7.69 (d, J=8.4Hz, 1H), 8.05 (s, 1H).

[0139] IV. Synthesis of 4′-O-phosphonooxymethyletoppside:

[0140] IVa. Synthesis of 4′-O-phosphonooxymethyletoposide dibenzylester:

[0141] To a solution of chloromethyl dibenzylphosphate (670 mg, 2.05mmol), etoposide (300 mg, 0.51 mmol), and tetrabutylammonium bromide(164.4 mg, 0.51 mmol) in tetrahydrofuran (0.5 mL) was added powderedpotassium carbonate (352.4 mg, 2.55 mmol). The resulting reactionmixture was vigorously stirred at room temperature for 35 minutes. Themixture was then directly purified by silica gel flash columnchromatography (30:1 methylene chloride/methanol) to give 272 mg (61%yield) of the title compound as a white solid with more than 95% of thetrans stereochemistry retained.

[0142] FABMS+(NBA): [MH]⁺, m/z 879.

[0143]¹H NMR (400 MHz, CDCl₃, δ): 1.41 (d, J=5.0 Hz, 3H), 2.79 (br s,1H), 2.86 (m, 1H), 2.97 (br s, 1H), 3.30 (dd, J=14.2, J=5.3 Hz, 1H),3.35 (m, 2H), 3.45 (t, J=8.5, J=8.0 Hz, 1H), 3.59 (m, 1H), 3.66 (s, 6H),3.74 (m, 1H), 4.19 (m, 1H), 4.20 (t, J=8.5, J=8.0 Hz, 1H), 4.42 (dd,J=10.3, J=9.1 Hz, 1H), 4.60 (d, J=5.2 Hz, 1H),4.64(d, J=7.6Hz, 1H), 4.76(q, J=5.0 Hz, 1H), 4.92 (d, J=3.4 Hz, 1H), 5.03 (dd, J=7.3, J=4.3 Hz,4H), 5.54 (dd, J=11.7, J=5.1 Hz, 1H), 5.59 (dd, J=11.3, J=5.1 Hz, 1H),5.99 (d, J =3.5 Hz, 2H), 6.26 (s, 2H), 6.51 (s, 1H), 6.84 (s, 1H), 7.33(m, 10H).

[0144]¹³C NMR (75 MHz, CDCl₃, δ): 20.21, 37.49, 41.00, 43.78, 56.07,66.32, 67.87, 67.97, 69.06, 69.14, 73.01, 73.29, 74.47, 79.70, 92.55,92.62, 99.70, 101.57, 101.72, 107.89, 109.13, 110.55, 127.82, 127.97,128.15, 128.35, 128.43, 132.40, 133.08, 135.68, 135.78, 136.49, 147.14,148.73, 152.18, 174.90.

[0145] IVb. Synthesis of 4′-O-phosphonooxymethyletoposide:

[0146] To a solution of 4′-O-phosphonooxymethyletoposide dibenzyl ester(20.5 mg, 0.023 mmol) in tetrahydrofuran (2 mL) was added palladium oncarbon (10%, 5 mg) . The mixture was stirred under an atmosphere ofhydrogen (1 atm) for 10 minutes. The catalyst was removed by filtrationthrough Celite, and tetrahydrofuran was evaporated at reduced pressure.The resulting residue was dried in vacuo to give 16 mg (100% yield) ofthe title compound as a white solid.

[0147] FABMS+(NBA) : [MH]⁺, m/z 699.

[0148]¹H NMR (400 MHz, CDCl₃/DMSO-d₆, δ): 1.29 (d, J=5.0 Hz, 3H), 2.78(m, 1H), 3.21 (m, 2H), 3.29 (t, J=8.6, J=7.8 Hz, 1H), 3.37 (dd, J=14.0,J=5.3 Hz, 1H), 3.52 (m, 2H), 3.62 (s, 6H), 4.09 (m, 1H), 4.17 (t, J=8.1Hz, 1H), 4.38 (dd, J=8.8, J=8.7 Hz, 1H), 4.44 (d, J=7.6 Hz, 1H), 4.48(d, J=5.3 Hz, 1H), 4.66 (q, J=5.0 Hz, 1H), 4.88 (d, J=3.3 Hz, 1H), 5.05(br s, 7H), 5.40 (dd, J=10.7, J=7.8 Hz, 1H), 5.43 (dd, J=10.4, J=7.5 Hz,1H), 5.89 (dd, J=8.8 Hz, 1H), 6.18 (s, 2H), 6.41 (s, 1H), 6.78 (s, 1H).

[0149] IVc. Synthesis of 4′-O-phosphonooxymethyletoposide disodium salt:

[0150] To a solution of 4′-O-phosphonooxymethyletoposide dibenzyl ester(200 mg, 0.227 mmol) in tetrahydrofuran (10 mL) was added palladium oncarbon (10%, 45 mg). This mixture was stirred under an atmosphere ofhydrogen (1 atm) for 25 minutes. The catalyst was removed by filtrationthrough Celite. The filtrate was evaporated at reduced pressure, and theresidue was dried in vacuo. The resulting white solid was dissolved inan aqueous solution of sodium bicarbonate (2.9 mL of 0.136 M=0.394mmol). The resulting heterogeneous mixture was mixed with activatedcarbon, stirred for a few minutes, and was then filtered through a 40 μmfilter unit. The homogenous, colorless filtrate was lyophilized to give140 mg (96% yield) of the title compound as a white solid with more than95% of bans stereochemistry retained.

[0151] FABMS+(NBA) : [MH]⁺, m/z 743, [M−Na+2H]⁺, m/z 721, [M−2Na+3H]⁺,m/z 699.

[0152]¹H NMR (400 MHz, D₂O, δ): 1.37 (d, J=5.1 Hz, 3H), 3.10 (m, 1H),3.37 (dd, J=8.9, J=8.0 Hz, 1H), 3.48 (m, 2H), 3.65 (m, 3H), 3.75 (s,6H), 4.29 (dd, J=10.4, J=4.5 Hz, 1H), 4.41 (t, J=8.3, J=8.0 Hz, 1H),4.49 (dd, J=10.5, J=8.9 Hz, 1H), 4.68 (d, J=5.7 Hz, 1H), 4.74 (d, J=7.8Hz, 1H), 4.91 (q, J=5.0 Hz, 1H), 5.13 (d, J=3.0 Hz, 1H), 5.26 (2xd,J=5.3, J=3.3 Hz, 1H), 5.28 (2xd, J=5.3, J=3.3 Hz, 1H), 5.98 (d, J=10.5Hz, 2H), 6.40 (s, 2H), 6.58 (s, 1H), 7.00 (s, 1H).

[0153]¹³C NMR (125 MHz, D₂O, δ): 22.13, 40.74, 43.56, 46.11, 59.12,68.70, 70.41, 72.40, 75.46, 75.95, 76.95, 82.46, 94.87, 102.88, 103.66,104.62, 111.14, 112.82, 113.23, 130.73, 135.45, 135.74, 140.22, 149.56,151.43, 154.94, 166.36, 181.61.

[0154]³¹P NMR (200 MHz, D2O, δ): s (2.19).

[0155] V. Synthesis of Phosphonooxymethylating Agents

[0156] Va. Synthesis of chloromethyldibenzyl phosphate

[0157] To a refluxed solution of chloroiodomethane (25 g of 97%, 0.14mol) in toluene (HPLC-grade, 30 mL) was added silver dibenzylphosphate(7.0 g, 0.018 mol) in several portions over 20 minutes. Refluxing wascontinued for one hour. After the reaction mixture was cooled down toroom temperature and filtered, the solvent was evaporated under reducedpressure. The oily residue was purified by silica gel flash columnchromatography (7:3 hexane/ethyl acetate) to give 3.63 g (62% yield) ofthe title compound as a yellow oil.

[0158] FABMS+(NBA): [MH]⁺, m/z 327

[0159]¹H NMR (300 MHz, CDCl₃, δ): 5.10 (d, J=8.0 Hz, 4H), 5.63 (d,J=15.7 Hz, 2H), 7.36 (s, 10H).

[0160]¹³C NMR (75 MHz, CDCl₃, δ): 69.68, 69.75, 73.33, 73.42, 127.93,128.51, 128.63, 135.07.

[0161] Vb. Synthesis of dibenzyl (p-toluenesulfonemethyl)-phosphate:

[0162] To a stirred solution of silver p-toluenesulfonate (600 mg, 2.15mmol) in dry acetonitrile (3 mL) was added chloromethyldibenzylphosphate (150 mg, 0.46 mmol) under an argon atmosphere. Afterthe reaction mixture was stirred for 21 hours at room temperature, thesolvent was removed, and the residue extracted with ether (3×3 mL). Thecombined extracts were filtered, evaporated, and dried in vacuo to give210 mg (99% yield) of the title compound as a white solid.

[0163] EIMS: [MH]⁺, m/z 463.

[0164]¹H NMR (300 MHz, CDCl₃, δ): 2.37 (s, 3H), 4.91 (2 x d, J=7.9 Hz,4H), 5.61 (d, J=14.2 Hz, 2H), 7.29 (m, 12H), 7.78 (d, J=8.4 Hz, 2H).

[0165] With respect to the above reaction Vb, as explained also in Icabove, the reagent:

[0166] can be generically represented by the following formula:

[0167] wherein all symbols are the same as defined above.

[0168] Vc. Synthesis of formaldehyde bis(dibenzyloxyphosphono)-acetal:

[0169] To a solution of diiodomethane (4 mL, 50 mmol) in dry toluene (15mL) was added silver dibenzylphosphate (3.0 g, 7.8 mmol). The resultingmixture was refluxed for 15 minutes under an argon atmosphere. Themixture was then cooled down to room temperature and filtered. Then thesolvent was evaporated in vacuo. The oily residue was purified by silicagel flash column chromatography (1:1 hexane/ethyl acetate and then ethylacetate) to yield a yellowish oil which then crystallized to give 1.97 g(90% yield) of the title compound as a white solid, mp 39-42° C.

[0170] CIMS (NH3): [MH]+, m/z 569.

[0171]¹H NMR (300 MHz, CDC₃, δ): 5.03 (d, J=7.9 Hz, 8H),5.49 (t, J=14.3Hz, 2H), 7.30 (m, 20H).

[0172]¹³C NMR (75 MHz, CDCl₃, δ): 69.54, 69.61, 86.48, 127.88, 128.48,128.55, 135.10, 135.20.

[0173] VI-Synthesis of O-Phosphonooxymethylcyclosporin A:

[0174] VIa. Synthesis of O-methylthiomethylcyclosporin A:

[0175] To a suspension of Cyclosporin A in dimethylsulfoxide (250 mL) isadded acetic anhydride (125 mL) and acetic acid (35 mL). Theheterogeneous mixture is vigorously stirred at room temperature for 24hours, poured into ice (800 mL), stirred for 30 minutes, and thenextracted with methylene chloride (4×100 mL). The combined methylenechloride extracts are washed with water (2×100 mL) and dried overmagnesium sulfate. The methylene chloride is removed at reduced pressureto provide a product. The product is further purified by silica gelchromatography.

[0176] VIb. Synthesis of O-phosphonooxymethylcyclosporin A dibenzylester:

[0177] To a well stirred suspension of O-methylthiomethylcyclosporin Aand powdered, activated 4 A molecular sieves (5 g) in tetrahydrofuran(20 mL) is added a suspension of N-iodosuccinimide (2.00 g of 95%, 8.44mmol) and dibenzylphosphate (2.20 g, 7.83 mmol) in methylene chloride(12 mL). The resulting mixture is vigorously stirred at room temperaturefor 30 minutes, filtered, and diluted with ethyl acetate (300 mL). Thesolution is washed with aqueous sodium thiosulfate (10%, 2×15 mL), water(2×20 mL), brine (50 mL), and dried over magnesium sulfate. The mixtureis filtered and the solvent is evaporated under reduced pressure. Theresidue is purified by silica gel flash column chromatography.

[0178] VIc. Synthesis of O-phosphonooxymethylcyclosporin A:

[0179] To a solution of Q-phosphonooxymethylcyclosporin A dibenzyl esterin tetrahydrofuran (100 mL) and water (5 mL) is added palladium oncarbon (10%, 500 mg). This mixture is stirred under an atmosphere ofhydrogen (1 atm) for 35 minutes. The catalyst is removed by filtrationthrough Celite. The Celite is then washed with tetrahydrofuran (300 mL)and the combined filtrates are evaporated at reduced pressure. Theresulting solid is washed with ether (2×20 mL), hexane (50 mL), dried invacuo, and then dissolved in hot methanol (60 mL). The solution isfiltered, concentrated at reduced pressure to ˜10 mL volume. Afterstanding at room temperature for one hour, the solution is placed in arefrigerator overnight. The crystalline precipitate that forms overnightis filtered off and dried in vacuo to give the title compound as asolid. The filtrate is concentrated to ˜1 mL volume and kept in therefrigerator for one hour to give additional product.

[0180] Biological Evaluation

[0181] Compounds of the present invention are novel pharmaceuticalagents; representative compounds of formula I have been evaluated in invitro and in vivo conversion studies. In all of these studies theprodrugs were converted into their pharmaceutically active parentcompounds.

[0182] (1) Solubility Estimate of Propofol Prodrug in Water

[0183] The water solubility of propofol prodrug is approximately 500mg/mL based on HPLC analysis of saturated aqueous solution.

[0184] (2) in vitro conversion of propofol prodrug to propofol

[0185] The in vitro conversion of propofol prodrug to propofol wasperformed using alkaline phosphatase in glycine buffer pH 10.4 medium.25 mL of a 100 μg/mL propofol prodrug solution in glycine buffer wasprepared. One millimeter was saved for a zero time point and theremaining 24 mL were placed in a 37° C. water bath. 960 μL of a 0.1mg/mL alkaline phosphatase in glycine buffer solution was added to the24 mL of propofol prodrug solution, mixed, and returned to the waterbath. 1.5 mL samples were removed at 5, 10, 20, 30, 40, 60, 90, 120,180, 240, 300, and 360 minutes. To each sample, 10 μL of glacial aceticacid was added immediately to stop the enzymatic reaction. The sampleswere assayed by HPLC to quantitate the propofol prodrug and propofolconcentration. The results of the in vitro conversion are shown inFIG. 1. These results demonstrate that the propofol prodrug is asubstrate for alkaline phosphatase.

[0186] (3) Gross Toxicity Evaluation in Rats

[0187] Propofol prodrug was prepared for i.v. injection at aconcentration of 68 mg/mL in 0.9% Sodium Chloride Injection, USP. Thisconcentration is equivalent to 36 mg/mL of propofol. The propofolprodrug solution was filtered through a 0.22 μm nylon membrane prior toadministration.

[0188] The evaluation of the propofol prodrug on rats was conducted withtwo male Harlen Sprague-Dawley rats weighing 820 and 650 g. The 820 grat received 200 μL of the propofol prodrug i.v. formulation (equivalentto 9 mg/kg of propofol) in the tail vein. A blood sample was taken fromthe tail vein (with heparinized syringe) after approximately 12 minutes.The 650 g rat received a dose of the mild sedative Metaphane® prior toreceiving the propofol prodrug formulation. The 650 g rat was injectedwith 125 μL of the propofol prodrug formulation in the tail vein and ablood sample was taken from the tail vein (with heparinized syringe)after approximately six minutes. The blood samples from both rats wereassayed for propofol by HPLC.

[0189] The results of the propofol prodrug injection in both rats weresimilar. Both rats became unsteady after a few minutes, but never losttheir righting reflex. Based on visual observations, the rats fullyrecovered from the propofol prodrug injections. Blood removed from bothrats confirmed the presence of propofol through HPLC analysis. The ratsdid not display signs of discomfort due to the propofol prodrug.

[0190] (4) Pharmacokinetic Evaluation in Dogs

[0191] A pharmacokinetic study involving Diprivan® or the propofolprodrug was performed in a dog with a sufficient washout period betweenstudies. The blood concentrations were determined using HPLC withfluorescence detection while brain activity was monitored with two leadelectroencephalography (EEG). Prior to dosing the dog, the dog wasblindfolded, cotton was placed in the ears of the dog, and the legs ofthe dog were bound to minimize movement and other outside stimuli sothat the effect of the propofol on the dog's brain wave activity couldbe most efficiently monitored.

[0192] The evaluation of the propofol blood concentration versus timewas conducted with a beagle weighing ˜13 kg. Approximately 8 mL of bloodwas taken prior to injection to be used for standard curve preparationand a zero time blood level. The dog received a volume of Diprivan® orpropofol prodrug formulation equivalent to 7 mg/kg of propofol viainjection in the cephalic vein.

[0193] Two mL blood samples were taken from either the cephalic (not thesame vein as the formulation injection site), jugular, or saphenous vein(with heparinized syringe) after 1, 3, 5, 10, 15, 20 and 30 minutesafter the injection. Blood samples were also taken after 60, 90, 120,180, 240, 300, 360, 480, and 1440 minutes. Blood samples were extractedto remove the propofol immediately after being taken from the dog. Thedog was fasted for approximately 20 hours prior to receiving theDiprivan® or propofol prodrug formulation. After the 120 minute samplewas taken, the dog was allowed to drink water. Food was given to the dogafter the 480 minute blood sample was obtained. The dog's regular dietwas Hills' Science Diet Maintenance. The dog was on a light/dark cycleof 12 hours of light per day.

[0194] The concentration of propofol in the blood samples was determinedusing HPLC with fluorescence detection. The results are shown in FIG. 2.The blood extraction and HPLC methods used were based on work reportedby Plummer (1987) with minor modifications. The sample preparation andassay procedure used were as follows:

[0195] To a 1 mL sample of blood, 10 μL of thymol internal standard (20μg/mL) and 1 mL phosphate buffer (0.1 M, pH 7.2) were added, vortexingto mix after each addition. Five mL of cyclohexane was then added andthe samples were mixed at 75 rpm for 20-30 minutes. The organic layerwas separated by 1 minute of centrifugation at approximately 2000 rmp.Approximately 4.5 mL of the organic layer was transferred to a tubecontaining 50 μL of dilute tetramethylammonium hydroxide (TMAH) solutionat, approximately 1.8% (w/v) . The solvent was evaporated to drynessunder a stream of nitrogen and reconstituted with 200 μL of Mobile PhaseA. The samples were centrifuged at 15,000 rmp for 30 seconds to removeany particles, and the supernatant was injected on the HPLC. Standardcurve samples were prepared by spiking 1 mL aliquots of the initialblood with propofol at concentrations 5, 1, 0.5, 0.1 and 0.01 μg/mL.These standards were treated the same as the samples.

[0196] The HPLC system consisted of the following Shimadzu components:LC-10AT pumps, SCL-10A system controller, RF 353 fluorescence detector,and SIL-10A auto sampler. The HPLC parameters were as follows:excitation at 275 nm and emission at 320 nm; flow rate at 1 mL/min;injection volume was 3-30 μL depending on propofol concentration. TheHPLC column was a Zorbax RX-C18, 15 cm×4.6 mm i.d., 5 μm particle size.Mobile Phase A was 60:40 (v/v) acetonitrile: 25 mM phosphate, 15 mM TBAPBuffer pH 7.1. Mobile Phase B was 80:10:10 (v/v/v)acetonitrile:water:THF. Mobile Phase B was used to clean the columnafter the thymol and propofol eluted using Mobile Phase A (4.2 and 7.4minutes, respectively).

[0197] The dog exhibited signs of anesthesia upon injection of bothformulations based on visual observations and EEG patterns. The dogrecovered from anesthesia from both formulations in 20-30 minutes.Propofol blood levels resulting from injection of the propofol prodrugapproximate those from injection of Diprivan®.

[0198] (5) Solubility Estimate of Camptothecin Prodrug in Water

[0199] The water solubility of the camptothecin prodrug is greater than50 mg/mL based on visual and HPLC analysis.

[0200] (6) Camptothecin Prodrug (p-cpt) Enzymatic Study

[0201] A 16 μg/mL p-cpt was cleaved with acid phosphatase (0.02 units/mLof p-cpt solution). The media was 0.09 M citrate buffer, pH 4.8 and thetemperature was 37° C. The conversion of p-cpt to camptothecin wasmonitored by HPLC.

[0202] HPLC Parameters:

[0203] MP: 24% potassium phosphate buffer pH 4, 76% acetonitrile

[0204] Column: Zorbax RX-CL8, 15 cm×4.6 mm i.d., 5 μm particle size

[0205] Detection: 370 nm UV

[0206] Flowrate: 1 mL/min

[0207] Acid phosphatase from bovine prostate (sigma). The results areshown in FIG. 3. The results demonstrate that the camptothecin prodrugis a substrate for phophatases.

[0208] (7) Pharmacokinetic Study of the Camptothecin Prodrug Using Rats

[0209] Pharmacokinetic experiments involving the dosing of maleSprague-Dawley rats with formulations of the camptothecin prodrug andcamptothecin were undertaken. The two formulations of the camptothecinprodrug that were examined consisted of the prodrug dissolved in 15 mMphosphate, pH 4.0 and camptothecin dissolved in organic co-solvents. Thefollowing is a summary of the pharmacokinetic experiments:

[0210] A volume of the camptothecin prodrug formulation or camptothecinformulation was prepared at a concentration so that a dose equivalent to1 mg camptothecin per kg weight could be given to the rat. Theformulation was given to the rat using an indwelling cannula in the leftjugular vein of the rat. Blood samples were taken via an indwellingcannula located in the right jugular vein of the rat. Both cannulas wererinsed with heparinized saline prior to use and contained heparinizedsaline during the study.

[0211] The rats were anesthetized with sodium pentobarbital prior toinsertion of the jugular cannulas and kept anesthetized with sodiumpentobarbital during the study. The rats were placed on a 37° C. heatingpad during the study and tracheotomized. Blood samples of approximately150 μL were taken prior to dosing and after 1, 3, 5, 10, 15, 20, 30, 45,60 and 90 minutes after the formulations were given to the rat.

[0212] The blood samples were placed in microcentrifuge tubes andcentrifuged for 20 seconds at approximately 15000 rpm. A 50 μL aliquotof plasma from each blood sample was transferred to a secondmicrocentrifuge tube. A 150 μL aliquot of chilled acetonitrile was addedto the plasma and the preparation is vortexed for 5 seconds. A 450 μLaliquot of chilled sodium phosphate (0.1 M, pH 7.2) was then added. Thecontents in the microcentrifuge tubes were vortexed for 5 seconds andcentrifuged for 20 seconds at approximately 15000 rpm. The supernatantwas transferred to an HPLC autosampler set at 4° C. and analyzed (50 μLinjections).

[0213] The HPLC system consisted of the following Shimadzu components:LC-10AT pump, SCL-10A system controller, RF 535 fluorescence detector,SIL-10A autosampler (set at 4° C.), and CTO-10A column oven (temperatureset at 30° C.). The HPLC parameters were as follows: excitation at 370nm and emission at 435 nm; flow rate at 2 mL/min. The HPLC column was aHypersil ODS, 15 cm×4.5 mm i.d., 5 μm particle size. The mobile phasewas 75% 25 nM sodium phosphate, pH 6.5/25% acetonitrile (v/v) with 25 mMtetrabutylammonium dihydrogen phosphate added as an ion-impairingreagent.

[0214] As can be seen in the graph (FIG. 4) the prodrug providescamptothecin plasma levels which are equivalent to those attained fromdirect injection of camptothecin in organic co-solvents. The graphprovides the mean with standard deviation for five rats which receivedprodrug and six rats which received camptothecin.

We claim:
 1. A compound according to formula I:

wherein, R—O— is a residue of n alcohol-containing or phenol-containingpharmaceutical compound, excluding taxol, R¹ is hydrogen or an alkalimetal ion or a protonated amine or a protonated amino acid, R² ishydrogen or an alkali metal ion or a protonated amine or a protonatedamino acid, and n is an integer of 1 or 2; and pharmaceuticallyacceptable salts thereof.
 2. The compound according to claim 1, whereinsaid alcohol-containing or phenol-containing compound is selected fromthe group consisting of camptothecin, camptothecin analogues, propofol,etoposide, vitamin E and cyclosporin A.
 3. The compound according toclaim 1, wherein the alkali metal ion of R¹ and R² is each independentlyselected from the group consisting of sodium, potassium and lithium. 4.A compound selected from the group consisting of:

wherein Z is selected from the group consisting of hydrogen, alkalimetal ion, and amine; and pharmaceutically acceptable salts thereof. 5.The compound according to claim 4, wherein each Z is independentlyselected from the group consisting of sodium, tromethamine,triethanolamine, triethylamine, arginine, lysine, ethanolamine andN-methylglucamine.
 6. A compound according to formula III:

wherein, R—O— is a residue of an alcohol-containing or phenol-containingpharmaceutical compound, excluding taxol; and pharmaceuticallyacceptable salts thereof.
 7. A compound according to claim 6, whereinsaid compound is selected from the group consisting of:


8. A compound according to formula IV:

wherein, R—O— is a residue of an alcohol-containing or phenol-containingpharmaceutical compound, excluding taxol, Y is a phosphono protectinggroup, and n is an integer of 1 or 2; and pharmaceutically acceptablesalts thereof.
 9. A compound according to claim 8, wherein said compoundis selected from the group consisting of:

wherein Y is a phosphono protecting group.
 10. The compound according toclaim 8, wherein said phosphono protecting group is selected from thegroup consisting of a benzyl group, a t-butyl group, an allyl group, andother acceptable phosphate protecting groups.
 11. A pharmaceuticalcomposition, comprising: an effective amount of a compound according toclaim 1; and a pharmaceutically acceptable carrier.
 12. A process forpreparing a compound of claim 4, comprising: removing a phosphonoprotecting group from a compound according to one of the followingformula:

wherein Y is the phosphono protecting group; and recovering the product.13. A process for preparing a compound of claim 6, comprising: reactinga compound of the formula R—O—H, wherein, R—O— is a residue of analcohol-containing or phenol-containing pharmaceutical compound,excluding taxol, and pharmaceutically acceptable salts thereof, withdimethylsulfoxide in the presence of acetic anhydride and acetic acid;and recovering the product.
 14. A process for preparing a compound ofclaim 7, comprising: reacting a compound according to formula III:

wherein, R—O— is a residue of an alcohol-containing or phenol-containingpharmaceutical compound, excluding taxol; and pharmaceuticallyacceptable salts thereof, with N-iodosuccinamide and a protectedphosphoric acid of formula HOP (O)(OY), wherein Y is a phosphonoprotecting group; and recovering the product.
 15. The process accordingto claim 14, wherein the phosphono protecting group is selected from thegroup consisting of a benzyl group, a t-butyl group and an allyl group.16. A method of treatment which comprises administering to a patient inneed thereof an effective amount of a composition according to claim 11.17. The method according to claim 16, wherein said compound isadministered orally.
 18. The method according to claim 16, wherein saidcompound is administered parenterally.